Project Summary Embryonic stem cells offer considerable potential for cell replacement therapy where cells have been lost to injury or disease because of their capacity for continual self renewal and ability to differentiate into virtually any cell type or tissue. Currently, however the process by which human embryonic stem cells (hESCs) differentiate into various mature functional cell types is poorly understood and there is a critical unmet need for methods of expanding and differentiating hESCs into desirable cell types such as epithelial, dermal, endothelial, cardiac and skeletal myocytes, etc. Scientists at Advanced Cell Technology have isolated hundreds of clonal cell populations (called lineage restricted cells, LRCs) derived from differentiating hESCs which appear to be both expandable in culture and have the properties of early progenitor cells of a multitude of mature cell types. We would like to study LRC differentiation in the context of a differentiating culture of hESCs where they receive signals from surrounding cells and matrix to better understand their therapeutic potential and how to direct their differentiation in culture. Near term, our goal is to identify specific targeting peptides for LRCs using phage display. Selection on these homogeneous progenitor populations will allow us to identify specific targeting peptides that target surface markers that might otherwise be under-represented when selecting on a hererogeneous population of differentiating hESCs. We will track the developmental fate of targeted LRCs in their native context of differentiating hESCs using quantum dot conjugated targeting peptides. These tools will allow use to study the differentiation of specific progenitor cells as it occurs in real time using time-lapse imaging. Multiplex labeling with 2 or more peptides will allow us to trace progenitor cell interactions during in vitro differentiation on hESCs. These studies will be used to select LRCs with high therapeutic potential and to develop methods for LRC differentiation. Our long term goal is to develop selected LRCs for use in regenerative therapies for skin, vasculature, and heart as well as commercialization of the LRCs and their peptide targeting agents as research reagents. . Project Narrative Embryonic stem cells offer considerable potential for cell replacement therapy where cells have been lost to injury or disease because of their capacity for continual self renewal and ability to differentiate into virtually any cell type or tissue. There is a critical unmet need for methods of expanding and differentiating hESCs in-vitro into desirable cell types such as skin, heart muscle, blood vessels, etc. We have isolated hundreds of pure lineage restricted cells (LRCs) derived from hESCs which are expandable in culture and have the properties of early progenitor cells of a multitude of mature cell types. Near term, our goal is to label specific LRCs with quantum dot labeled targeting agents so that we can track the developmental fate of LRCs in cultures of differentiating hESCs and thus determine their therapeutic potential. Our long term goal is to develop selected LRCs for use in regenerative therapies for skin, vasculature, and heart among others as well as commercialization of the LRCs and their peptide targeting agents as research reagents.